Rosita Zakaria and Ronda F. Greaves ORCID logo

The re-emergence of dried blood spot sampling – are we ready?

De Gruyter | Published online: October 30, 2019

“Everything old is new again” is an appropriate description for the recent re-emergence of dried blood spot (DBS)-based methods outside of the traditional realm of newborn screening. From its humble beginnings over a century ago [1], DBS sampling emerged 50 years later with the worldwide application of capillary sampling of babies for newborn bloodspot screening (NBS) [2], [3]. Stepping forward another 50 years, we now see an increase in translational research publications for dried matrices (primarily blood, but also urine, saliva and sweat) looking for their place as mainstream applications [4]. Whilst there are significant drivers for microsampling with DBS, we need to consider whether these emerging applications achieve appropriate quality goals and traceability to ensure they are fit for their clinical purpose [5], [6].

In this issue of Clinical Chemistry and Laboratory Medicine (CCLM), there are three timely articles highlighting the utility and continued challenges of DBS analysis [7], [8], [9]. The articles clearly demonstrate the emergence and growth of DBS applications from research to mainstream application and together they highlight specific considerations across the total testing process of the brain-to-brain loop [10]. The first article discusses at improving the pre-analytical process through the development of a phone-based application (app) for timely determination of DBS quality [8]. The other two articles discuss analytical considerations for mass spectrometry-based DBS methods and examine the post-analytical components by comparing plasma with DBS result interpretation [7], [9].

Pre-analytical errors associated with poor spot quality, which is a contributor to inaccurate results, can be high. The quality of the spot collection, even in controlled environments with experienced phlebotomists, can lead to a 4%–5% rejection rate [11]. This rejection rate can escalate to 20% for paediatric home-based sampling [12]. Even 4% is high compared to NBS, which has an achievable rejection rate guideline of less than 2% [13]. Hence, a lower error rate for these emerging applications is potentially achievable.

The report by Veenhof and colleagues describes a Dried Blood Spot Photo App that has been developed to determine if the quality of the DBS is acceptable or should be rejected (and therefore re-collected) prior to the sample being sent to the laboratory [8]. The app is reported to detect the size and overall appearance of the blood spot, but it does not detect haemolysis or humidity errors. The app was tested on the Apple IPhone 5s with an eight-megapixel camera in the laboratory with standard fluorescent lighting. Previously, spot quality was decided once the sample reached the laboratory. This retrospective quality check results in significant inconvenience for the patient and a delay in time for analysis. Although the current version of this app does not claim to detect all the possible pre-analytical errors associated with DBSs, it does provide an immediate indication as to whether a sufficient sample has been collected. In addition to the authors’ proposed use of the app, it can potentially also be utilised for training and education to improve the analytical quality.

The continued emergence of improved sensitivity in mass spectrometry-based technologies has underpinned the recent rapid expansion in the availability of applications for microsampling [4]. Clinicians and consumers are driving the demand for this technology with the laboratory professional the guardian of performance quality. This is highlighted by both Polo and Veenhof in their respective decisions on acceptability of the methods for clinical use [7], [9]. Whilst the plasma sphingolipid method is described as the “gold standard”, the DBS was described as “a useful tool” and considered acceptable for diagnosis. On the other hand, the transplant drug method was considered inadequate in the “strict clinical setting” for monitoring. Interestingly, whilst the authors apply the same decision and acceptance criteria for both plasma and DBSs, the method validation criteria presented are different between these two publications, the description of which ideally could be standardised [14] (Table 1).

Table 1:

Comparison of information provided for the discussed DBS mass spectrometry methods.

Method Veenhof et al. [7] Polo et al. [9] Millington et al. [3]
Pre-analytical
 Subjects Adults (app. n=40) Adults and paediatrics (app. n=114) Paediatrics (n=NS)
 Specimen type Venous whole blood and capillary finger prick DBS Venous whole blood and capillary finger prick DBS NBS cards
 DBS collection Finger prick/capillary Finger prick/capillary OR 50 μL venous whole blood spotted on the filter paper Heel prick/capillary
 Storage Whole blood <24 h, DBS <74 h ambient/DBS >29 h at −20 °C Plasma at −80 °C, DBS at −20 °C Ambient
Analytical
 Haematocrit correction No No No
 Punch size/location 8 mm/Central 3.2 mm/? Whole spot/NS
 Measurement LC-MS/MS LC-MS/MS FABa-MS/MS
 Calibrator matrix Dried spot Dried spot NS
Method validation
 Overall correlation R2=0.93–0.97 R2=0.59–0.98 NS
 Overall difference Positive bias Positive bias (except for LysoGb3) NS
 FDA/EMA acceptance criteria (67%±20%) NA NS
 Limits of clinical relevance (85%–115%) criteria >80% NA NS
 QCs relative error (accuracy) NA 75%–119% NS
 Median predicted percentage error criteria <15% NA NA
 Receiver-operating characteristic curve NA Plasma AUCb>DBS AUC NA
Post-analytical
 Suggested conversion factor NA e.g. 2.7 NA
 Primary purpose of the method Diagnosis Monitoring Population screening
 FFPc (Authors’ discussion) Rejected Accepted Accepted

    For comparison, the original DBS mass spectrometry method for NBS of amino acids and acyl carnitines is provided. aFAB, fast atom bombardment; bAUC, area under the curve; cFFP, fitness for purpose; NA, not applicable. h, hours; QC, quality control; FDA, US Food and Drug Administration; EMA, European Medicines Agency; NS, not specified.

Many consumers maintain an interest in DBS testing due to a desire to improve the long-term monitoring of particular health conditions. Whilst it is clear that the agreement between the plasma and DBS matrix is not ideal, with the DBS described as “not quite as good as plasma in terms of discrimination” for the sphingolipid methods and “not good enough” for the transplant drugs, neither of the studies evaluated the intra-patient agreement longitudinally. This may be of value as there is evidence to suggest that the correlation between the DBS and plasma improves for repeat sampling within the one patient [15]. This is likely due, at least in part, to consistent haematocrit and extraction efficiency of the DBS sample. Hence, there is a potential need to reconsider the way in which we evaluate DBS methods moving forward.

Similar to the available pre-analytical DBS guideline, there is a need for DBS-specific method validation guidance [16]. For accuracy of quantitation, the method validation of DBS requires additional considerations over conventional liquid blood testing. This includes the influence of: (1) haematocrit and blood diffusion pattern; (2) punch location; (3) extraction efficiency; (4) traceability of the method; and (5) application of correction factors [17]. In addition, where the comparison is between spotted or liquid venous blood and a capillary DBS, the probable difference between capillary and venous levels of the target metabolite needs to be considered. The final part of the method validation that requires close consideration relates to the decision limits used and how they relate to the liquid matrix if applicable. Together with the development of a specific guideline, it will be the future availability of matrix-matched commercially available traceable reference materials for calibration of the assays and external quality assurance programs that will significantly enhance the harmonisation and standardisation of the DBS analysis [16], [18].

In summary, the three manuscripts in this addition of CCLM highlight the emergence of DBS as a standard matrix for testing. With this, the hybrid laboratory model, which facilitate consumer access, is likely to grow for mass spectrometry and other methods based on DBS microsampling [5], [19]. Part of the adjustment to the changing landscape will be a review of how we manage the quality of the total testing process (including method validation procedures) to ensure the results generated are fit for their clinical purpose.

Acknowledgments

We wish to thank Mr Ian Farrance and Ms Britta Maunder from Australia for critically reading this manuscript prior to submission. A/Prof Greaves is an officer of the IFCC-Emerging Technologies Division – https://www.ifcc.org/ifcc-emerging-technologies-division/.

References

1. Bang I. A Micro Determination of Blood Components [Ein verfahren zur mikrobestimmung von blutbestandteilen]. Biochem Ztschr 1913;49:19–39. Search in Google Scholar

2. Guthrie R, Susi A. A simple phenyalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 1963;32:338–43. Search in Google Scholar

3. Millington DS, Kodo N, Norwood DL, Roe CR. Tandem mass spectrometry: a new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism. J Inherit Metab Dis. 1990;13:321–4. Search in Google Scholar

4. Zakaria R, Allen KJ, Koplin JJ, Roche P, Greaves RF. Advantages and challenges of dried blood spot analysis by mass spectrometry across the total testing process. EJIFCC 2016;27:288–317. Search in Google Scholar

5. Plebani M. The future of laboratory medicine: navigating between technology and professionalism. Clin Chim Acta 2019;498:16. Search in Google Scholar

6. Greaves RF, Bernardini S, Ferrari M, Fortina P, Gouget B, Gruson D, et al. Key questions about the future of laboratory medicine in the next decade of the 21st century: a report from the IFCC-Emerging Technologies Division. Clin Chim Acta 2019;495:570–89. Search in Google Scholar

7. Veenhof H, Koster RA, Alffenaar JC, van den Berg AP, de Groot MR, Verschuuren EA, et al. Clinical application of a dried blood spot assay for sirolimus and everolimus in transplant patients. Clin Chem Lab Med 2019;57:1854–62. Search in Google Scholar

8. Veenhof H, Koster RA, Brinkman R, Senturk E, Bakker SJ, Berger SP, et al. Performance of a web-based application measuring spot quality in dried blood spot sampling. Clin Chem Lab Med 2019;1846–53. Search in Google Scholar

9. Polo G, Burlina AP, Ranieri E, Colucci F, Rubert L, Pascarella A, et al. Plasma and dried blood spot lysosphingolipids for the diagnosis of different sphingolipidoses: a comparative study. Clin Chem Lab Med 2019;57:1863–74. Search in Google Scholar

10. Plebani M, Laposata M, Lundberg GD. The brain-to-brain loop concept for laboratory testing 40 years after its introduction. Am J Clin Pathol 2011;136:829–33. Search in Google Scholar

11. Veenhof H, Koster RA, Alffenaar JC, Berger SP, Bakker SJ, Touw DJ. Clinical validation of simultaneous analysis of tacrolimus, cyclosporine A, and creatinine in dried blood spots in kidney transplant patients. Transplantation 2017;101:1727–33. Search in Google Scholar

12. Al-Uzri A, Freeman KA, Wade J, Clark K, Bleyle LA, Munar M, et al. Longitudinal study on the use of dried blood spots for home monitoring in children after kidney transplantation. Pediatr Transplant 2017;21. Search in Google Scholar

13. Human Genetics Society of Australasia. Newborn bloodspot testing. Available from: https://www.hgsa.org.au/documents/item/29, 2011. Search in Google Scholar

14. Vogeser M, Schuster C, Rockwood AL. A proposal to standardize the description of LC–MS-based measurement methods in laboratory medicine. Clinical Mass Spectrometry. 2019;13:36–8. Search in Google Scholar

15. Groselj U, Murko S, Zerjav Tansek M, Kovac J, Trampus Bakija A, Repic Lampret B, et al. Comparison of tandem mass spectrometry and amino acid analyzer for phenylalanine and tyrosine monitoring – implications for clinical management of patients with hyperphenylalaninemia. Clin Biochem 2015;48:14–8. Search in Google Scholar

16. CLSI. Blood collection on filter paper for newborn screening programs, approved Standard – Sixth Edition. CLSI Document NBS01-A6. Wayne, PA: Clinical Laboratory Standards Institute, 2013. Search in Google Scholar

17. Zakaria R, Allen KJ, Koplin JJ, Roche P, Greaves RF. Candidate reference method for determination of vitamin D from dried blood spot samples. Clin Chem Lab Med 2019. DOI: https://doi.org/10.1515/cclm-2019-0397. Search in Google Scholar

18. Plebani M. Harmonization in laboratory medicine: the complete picture. Clin Chem Lab Med 2013;51:741–51. Search in Google Scholar

19. Phillips KA, Trosman JR, Douglas MP. Emergence of hybrid models of genetic testing beyond direct-to-consumer or traditional labs. J Am Med Assoc 2019;321:2403–4. Search in Google Scholar

Published Online: 2019-10-30
Published in Print: 2019-11-26

©2019 Walter de Gruyter GmbH, Berlin/Boston